PROJECT SUMMARY/ABSTRACT Title: Heme allocation and disruptions in asthma and the failing heart The candidate, Dr. Sweeny, is a postdoctoral fellow dedicated to developing a successful independent research career investigating heme homeostasis and allocation in healthy and disease states with a specific focus on key proteins and small molecule stimuli. With a strong background in biochemistry, biophysics, and molecular biology, the candidate has developed new expertise in cell biology techniques and will use the mentored phase of this award to move into translational research. The Career Development Plan outlined in this proposal covers two years of mentored training including technical skill training, career development activities, and guidance by an excellent advisory committee to facilitate the successful transition to independence. Research Plan: Heme proteins play a role in a number of crucial cellular processes, however, free heme is highly toxic and therefore its production, degradation and delivery must be tightly regulated. Heme delivery and insertion into heme proteins is essential for their function, and reallocation of heme in response to stimuli is crucial to maintain normal cellular activity, nevertheless, details of the heme homeostasis system and directed delivery upon stimuli remain elusive. The overarching goal of this research is to understand how heme homeostasis and heme allocation are controlled and regulated in cells, and to determine disruptions to these pathways in disease. Recent findings have identified three major players in heme sequestration, delivery and insertion into downstream proteins; GAPDH, nitric oxide (NO) and Hsp90. In Aim 1, during the mentored phase of the award, Dr. Sweeny will elucidate how NO and Hsp90 stimulate insertion of GAPDH-bound heme into the downstream heme proteins soluble guanylyl cyclase (sGC) and NADPH oxidase 5 (NOX5). Both sGC and NOX5 are implicated in a number of human diseases including asthma and cardiovascular disorders. This aim will be primarily carried out in model mammalian cell systems, supported by in vitro biochemical and biophysical assays, by investigating the interplay of Hsp90 and GAPDH, the effect of NO on GAPDH heme related activities, and determining the role of STIP1/HOP in the heme transfer machinery. In Aim 2, Dr. Sweeny will study the regulation of these downstream heme proteins in the context of human disease; asthma (sGC) and atrial fibrillation (AF) (NOX5). Aim 2A experiments will be carried out using airway smooth muscle cells from patients with severe asthma and age/gender/race matched controls. Aim 2B focuses on studying NOX5 regulation and contribution to disease in AF. This work will be carried out using tissue from AF patients undergoing cardiac surgery, and from donor hearts in sinus rhythm, and using cardiomyocytes differentiated from patient derived induced pluripotent stem cells. Aim 2B, translational research in heme homeostasis and allocation in the heart, extends beyond the scope of the mentor's lab and will be carried into the independent phase of the award. This application builds upon strong preliminary data, a supportive research environment, and an advisory committee with nationally recognized leaders in cardiopulmonary research.